1. Field of the Invention
The present invention relates to a novel cholesterol oxidase having stability in the presence of surfactant and a gene encoding the novel cholesterol oxidase.
2. Brief Description of the Background Art
A cholesterol oxidase is an oxidase which catalyses the reaction between 3β-hydroxysteroid and oxygen to thereby form the corresponding 3-oxosteroid and hydrogen peroxide. Its research and development have so far been made for the purpose of applying it to the measurement of cholesterol concentration in body fluids (e.g., JP-A-6-169765), production of cholesterol derivatives (e.g., JP-A-6-113883), insecticides (e.g., U.S. Pat. No. 5,558,862), detergents (e.g., WO 89/09813) and the like.
It is known that the enzyme is produced by many genera of microorganisms such as Streptomyces (e.g., JP-A-62-285789 (corresponding to EP-A-0560983)), Brevibacterium (e.g., JP-A4-218367 (corresponding to EP-A-0452112)), Rhodococcus (e.g., JP-T-3-503487 (corresponding to WO 90/05788)) and Pseudomonas (e.g., JP-A-6-189754).
The cholesterol oxidase isolated from Burkholderia cepacia ST-200 (Pseudonmnas sp. ST-200 according to the old classification, has been deposited on Feb. 4, 1998 in National Institute of Bioscience and Human Technology (NIBH), Ministry of International Trade and Industry, (1-3, Higashi 1-chome, Tsukuba-shi, Ibarki, Japan), its deposit number is FERM MP-6661) (e.g., Japanese Patent No. 3,241,712 (corresponding to US-A-2003/153051)) is characterized in that its product when cholesterol is used as the substrate is different, its reaction mechanism is different and it has high organic solvent resistance and temperature stability, in comparison with other cholesterol oxidase so far known. Since a gene of the cholesterol oxidase derived from Burkholderia cepacia ST-200 has been cloned, high level production of a recombinant cholesterol oxidase can be carried out in various hosts through its secretion or intracellular accumulation (e.g., JP-A-2002-65271).
The characteristics required for an enzyme to be used in an agent for clinical diagnosis are, in most cases, high reactivity and specificity to the object and thermal stability which shows influence upon preservation stability of the product, but in the case of the measurement of cholesterol, a surfactant is frequently prescribed at a high concentration for the purpose of measuring the object at a high specificity, so that in addition to the above-described characteristics, a preservation stability in the coexistence of a surfactant is also strongly in demand for the enzyme to be used. Although the cholesterol oxidase derived from Burkholderia cepacia ST-200 has the above-described excellent properties, this enzyme is inferior in terms of the preservation stability in the coexistence of a surfactant, and therefore it is difficult to apply the enzyme to a cholesterol measuring reagent.
Regarding the surfactant to be used in the cholesterol measuring system, 1-pentanesulfonate, 1-hexanesulfonate, 1-heptanesulfonate, 1-octanesulfonate, polyoxyethylene alkyl ether sulfate, sodium dodecylbenzenesulfonate, a cholic acid salt (sodium cholate), cholic acid, dehydrocholate, deoxycholic acid, sodium deoxycholate, sodium taurodeoxycholate, N,N-bis(3-D-gluconamidopropyl)cholamide, N,N-bis-3-D-gluconamidopropylcholamide, dodecylbenzenesulfonate, lauroylsarcosine and the like are used as anionic surfactants (e.g., JP-A-8-116996, Japanese Patent No. 2,799,835 (corresponding to JP-A-7-13607), JP-A-11-56395, JP-A-2000-60600 (corresponding to EP-A-0964249), JP-A-2002-142799 (corresponding to EP-A-1342792), Japanese Patent No. 3,529,081 (corresponding to JP-A-10-232219), JP-A-10-84997 (corresponding to EP-A-0821239)). In addition, n-dodecyltrimethylammonium chloride, hexadecylpyridinium chloride and the like are used as cationic surfactants (e.g., JP-A-10-84997 (corresponding to EP-A-0821239)).
As nonionic surfactants, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene-polyoxypropylene condensate, acyl polyoxyethylene sorbitan ester, alkyl polyoxyethylene ether, n-dodecyl-β-D-maltoside, sucrose monolaurate, polyoxyethylene lauryl ether, polyoxyethylene alkylene phenyl ether, polyoxyethylene alkylene tribenzyl phenyl ether, polyoxyethylene glycol p-t-octyl phenyl ether, polyoxyethylene higher alcohol ether, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene alkylamine, glycerol fatty acid ester, n-octyl-β-D-thioglucoside, cetyl ether (C16), lauryl ether (C12), oleyl ether, behenyl ether (C20), polyoxyethylene monolaurate and the like are used (e.g., Japanese Patent No. 2,799,835 (corresponding to JP-A-7-13607), JP-A-11-56395, JP-A-2000-60600 (corresponding to EP-A-0964249), JP-A-2002-142799 (corresponding to EP-A-1342792), Japanese Patent No. 3,529,081 (corresponding to JP-A-10-232219), JP-A-10-84997 (corresponding to, EP-A-0821239), JP-A-2000-325097, JP-A-2001-124780, JP-A-2000-116400, JP-A-9-299, JP-A-2001-346598, JP-A-9-224697, Japanese Patent No. 3,193,634 (corresponding to JP-A-9-313200), JP-A-10-210999).
In addition, betaine derivatives, alkylbetaine derivatives, imidazoliumbetaine derivatives, sulfobetaine derivatives, aminocarboxylic acid derivatives, imidazoline derivatives, amine oxanoide derivatives, bile acid derivatives and the like are used as ampholytic surfactants (e.g., JP-A-2000-60600 (corresponding to EP-A-0964249), JP-A-10-84997 (corresponding to EP-A-0821239), JP-A-2000-325097, JP-A-2001-124780).